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1.
Mol Ther ; 27(4): 773-784, 2019 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-30885573

RESUMO

First attempts to use exogenous mRNA for protein expression in vivo were made more than 25 years ago. However, widespread appreciation of in vitro transcribed mRNA as a powerful technology for supplying therapeutic proteins to the body has evolved only during the past few years. Various approaches to turning mRNA into a potent therapeutic have been developed. All of them share utilization of specifically designed, rather than endogenous, sequences and thorough purification protocols. Apart from this, there are two fundamental philosophies, one promoting the use of chemically modified nucleotides, the other advocating restriction to unmodified building blocks. Meanwhile, both strategies have received broad support by successful mRNA-based protein treatments in animal models. For such in vivo use, specifically optimized mRNA had to be combined with potent formulations to enable efficient in vivo delivery. The present review analyzes the applicability of mRNA technology to antibody therapy in all main fields: antitoxins, infectious diseases, and oncology.


Assuntos
Anticorpos Monoclonais/genética , Sistemas de Liberação de Medicamentos/métodos , Imunização Passiva/métodos , RNA Mensageiro/administração & dosagem , RNA Mensageiro/uso terapêutico , Animais , Doenças Transmissíveis/terapia , Composição de Medicamentos/métodos , Humanos , Lipídeos/química , Nanopartículas/química , Neoplasias/terapia , Toxinas Biológicas/imunologia
2.
Cancer Immunol Immunother ; 68(5): 799-812, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30770959

RESUMO

CV9201 is an RNActive®-based cancer immunotherapy encoding five non-small cell lung cancer-antigens: New York esophageal squamous cell carcinoma-1, melanoma antigen family C1/C2, survivin, and trophoblast glycoprotein. In a phase I/IIa dose-escalation trial, 46 patients with locally advanced (n = 7) or metastatic (n = 39) NSCLC and at least stable disease after first-line treatment received five intradermal CV9201 injections (400-1600 µg of mRNA). The primary objective of the trial was to assess safety. Secondary objectives included assessment of antibody and ex vivo T cell responses against the five antigens, and changes in immune cell populations. All CV9201 dose levels were well-tolerated and the recommended dose for phase IIa was 1600 µg. Most AEs were mild-to-moderate injection site reactions and flu-like symptoms. Three (7%) patients had grade 3 related AEs. No related grade 4/5 or related serious AEs occurred. In phase IIa, antigen-specific immune responses against ≥ 1 antigen were detected in 63% of evaluable patients after treatment. The frequency of activated IgD+CD38hi B cells increased > twofold in 18/30 (60%) evaluable patients. 9/29 (31%) evaluable patients in phase IIa had stable disease and 20/29 (69%) had progressive disease. Median progression-free and overall survival were 5.0 months (95% CI 1.8-6.3) and 10.8 months (8.1-16.7) from first administration, respectively. Two- and 3-year survival rates were 26.7% and 20.7%, respectively. CV9201 was well-tolerated and immune responses could be detected after treatment supporting further clinical investigation.


Assuntos
Linfócitos B/imunologia , Vacinas Anticâncer/imunologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Imunoterapia/métodos , Neoplasias Pulmonares/terapia , RNA Mensageiro/uso terapêutico , Linfócitos T/imunologia , Idoso , Idoso de 80 Anos ou mais , Antígenos de Neoplasias/genética , Vacinas Anticâncer/genética , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Células Cultivadas , Feminino , Humanos , Imunoterapia/efeitos adversos , Reação no Local da Injeção/etiologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/mortalidade , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , RNA Mensageiro/administração & dosagem , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Análise de Sobrevida
3.
J Immunol ; 198(4): 1595-1605, 2017 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-28077601

RESUMO

Among innovative adjuvants conferring a Th1-shift, RNAdjuvant is a promising candidate. This adjuvant consists of a 547-nt uncapped noncoding ssRNA containing polyU repeats that is stabilized by a cationic carrier peptide. Whereas vaccination of mice with an influenza subunit vaccine induced moderate virus-specific IgG1, vaccination together with RNAdjuvant significantly enhanced this IgG1 and additionally promoted the formation of IgG2b/c, which is indicative of Th1 responses. Furthermore, such sera neutralized influenza virus, whereas this effect was not detected upon vaccination with the subunit vaccine alone. Similarly, upon vaccination with virus-like particles displaying vesicular stomatitis virus G protein, RNAdjuvant promoted the formation of virus-specific IgG2b/c and enhanced neutralizing IgG responses to an extent that mice were protected against lethal virus infection. RNAdjuvant induced dendritic cells to upregulate activation markers and produce IFN-I. Although these effects were strictly TLR7 dependent, RNAdjuvant-mediated augmentation of vaccine responses needed concurrent TLR and RIG-I-like helicase signaling. This was indicated by the absence of the adjuvant effect in vaccinated MyD88-/-Cardif-/- mice, which are devoid of TLR (with the exception of TLR3) and RIG-I-like helicase signaling, whereas in vaccinated MyD88-/- mice the adjuvant effect was reduced. Notably, i.m. RNAdjuvant injection induced local IFN-I responses and did not induce systemic effects, implying good tolerability and a favorable safety profile for RNAdjuvant.


Assuntos
Adjuvantes Imunológicos , Imunoglobulina G/sangue , Vacinas contra Influenza/imunologia , Glicoproteínas de Membrana/imunologia , RNA não Traduzido/imunologia , Receptor 7 Toll-Like/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Adjuvantes Imunológicos/efeitos adversos , Animais , Anticorpos Antivirais/sangue , Proteína DEAD-box 58/imunologia , Proteína DEAD-box 58/metabolismo , Imunoglobulina G/imunologia , Vacinas contra Influenza/administração & dosagem , Glicoproteínas de Membrana/administração & dosagem , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Fator 88 de Diferenciação Mieloide/deficiência , Fator 88 de Diferenciação Mieloide/metabolismo , Células Th1/imunologia , Receptor 7 Toll-Like/metabolismo , Vacinação , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Proteínas do Envelope Viral/administração & dosagem , Proteínas do Envelope Viral/imunologia
4.
Lancet ; 390(10101): 1511-1520, 2017 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-28754494

RESUMO

BACKGROUND: Vaccines based on mRNA coding for antigens have been shown to be safe and immunogenic in preclinical models. We aimed to report results of the first-in-human proof-of-concept clinical trial in healthy adults of a prophylactic mRNA-based vaccine encoding rabies virus glycoprotein (CV7201). METHODS: We did an open-label, uncontrolled, prospective, phase 1 clinical trial at one centre in Munich, Germany. Healthy male and female volunteers (aged 18-40 years) with no history of rabies vaccination were sequentially enrolled. They received three doses of CV7201 intradermally or intramuscularly by needle-syringe or one of three needle-free devices. Escalating doses were given to subsequent cohorts, and one cohort received a booster dose after 1 year. The primary endpoint was safety and tolerability. The secondary endpoint was to determine the lowest dose of CV7201 to elicit rabies virus neutralising titres equal to or greater than the WHO-specified protective antibody titre of 0·5 IU/mL. The study is continuing for long-term safety and immunogenicity follow-up. This trial is registered with ClinicalTrials.gov, number NCT02241135. FINDINGS: Between Oct 21, 2013, and Jan 11, 2016, we enrolled and vaccinated 101 participants with 306 doses of mRNA (80-640 µg) by needle-syringe (18 intradermally and 24 intramuscularly) or needle-free devices (46 intradermally and 13 intramuscularly). In the 7 days post vaccination, 60 (94%) of 64 intradermally vaccinated participants and 36 (97%) of 37 intramuscularly vaccinated participants reported solicited injection site reactions, and 50 (78%) of 64 intradermally vaccinated participants and 29 (78%) of 37 intramuscularly vaccinated participants reported solicited systemic adverse events, including ten grade 3 events. One unexpected, possibly related, serious adverse reaction that occurred 7 days after a 640 µg intramuscular dose resolved without sequelae. mRNA vaccination by needle-free intradermal or intramuscular device injection induced virus neutralising antibody titres of 0·5 IU/mL or more across dose levels and schedules in 32 (71%) of 45 participants given 80 µg or 160 µg CV7201 doses intradermally and six (46%) of 13 participants given 200 µg or 400 µg CV7201 doses intramuscularly. 1 year later, eight (57%) of 14 participants boosted with an 80 µg needle-free intradermal dose of CV7201 achieved titres of 0·5 IU/mL or more. Conversely, intradermal or intramuscular needle-syringe injection was ineffective, with only one participant (who received 320 µg intradermally) showing a detectable immune response. INTERPRETATION: This first-ever demonstration in human beings shows that a prophylactic mRNA-based candidate vaccine can induce boostable functional antibodies against a viral antigen when administered with a needle-free device, although not when injected by a needle-syringe. The vaccine was generally safe with a reasonable tolerability profile. FUNDING: CureVac AG.


Assuntos
Imunogenicidade da Vacina , RNA Mensageiro/imunologia , Vacina Antirrábica/administração & dosagem , Raiva/prevenção & controle , Adolescente , Adulto , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Método Duplo-Cego , Vias de Administração de Medicamentos , Esquema de Medicação , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Feminino , Alemanha , Humanos , Masculino , Estudos Prospectivos , Vacina Antirrábica/imunologia , Adulto Jovem
5.
Cancer Immunol Immunother ; 67(4): 653-662, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29335856

RESUMO

BACKGROUND: Tumor metastasis and immune evasion present major challenges of cancer treatment. Radiotherapy can overcome immunosuppressive tumor microenvironments. Anecdotal reports suggest abscopal anti-tumor immune responses. This study assesses abscopal effects of radiotherapy in combination with mRNA-based cancer vaccination (RNActive®). METHODS: C57BL/6 mice were injected with ovalbumin-expressing thymoma cells into the right hind leg (primary tumor) and left flank (secondary tumor) with a delay of 4 days. Primary tumors were irradiated with 3 × 2 Gy, while secondary tumors were shielded. RNA and combined treatment groups received mRNA-based RNActive® vaccination. RESULTS: Radiotherapy and combined radioimmunotherapy significantly delayed primary tumor growth with a tumor control in 15 and 53% of mice, respectively. In small secondary tumors, radioimmunotherapy significantly slowed growth rate compared to vaccination (p = 0.002) and control groups (p = 0.01). Cytokine microarray analysis of secondary tumors showed changes in the cytokine microenvironment, even in the non-irradiated contralateral tumors after combination treatment. CONCLUSION: Combined irradiation and immunotherapy is able to induce abscopal responses, even with low, normofractionated radiation doses. Thus, the combination of mRNA-based vaccination with irradiation might be an effective regimen to induce systemic anti-tumor immunity.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Modelos Animais de Doenças , Ovalbumina/imunologia , RNA Mensageiro/imunologia , Radioimunoterapia , Timoma/terapia , Neoplasias do Timo/terapia , Animais , Terapia Combinada , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/genética , RNA Mensageiro/genética , Timoma/genética , Timoma/imunologia , Neoplasias do Timo/genética , Neoplasias do Timo/imunologia
6.
J Transl Med ; 15(1): 1, 2017 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-28049494

RESUMO

BACKGROUND: Prophylactic and therapeutic vaccines often depend upon a strong activation of the innate immune system to drive a potent adaptive immune response, often mediated by a strong adjuvant. For a number of adjuvants immunological readouts may not be consistent across species. METHODS: In this study, we evaluated the innate immunostimulatory potential of mRNA vaccines in both humans and mice, using a novel mRNA-based vaccine encoding influenza A hemagglutinin of the pandemic strain H1N1pdm09 as a model. This evaluation was performed using an in vitro model of human innate immunity and in vivo in mice after intradermal injection. RESULTS: Results suggest that immunostimulation from the mRNA vaccine in humans is similar to that in mice and acts through cellular RNA sensors, with genes for RLRs [ddx58 (RIG-1) and ifih1 (MDA-5)], TLRs (tlr3, tlr7, and tlr8-human only), and CLRs (clec4gp1, clec2d, cledl1) all significantly up-regulated by the mRNA vaccine. The up-regulation of TLR8 and TLR7 points to the involvement of both mDCs and pDCs in the response to the mRNA vaccine in humans. In both humans and mice activation of these pathways drove maturation and activation of immune cells as well as production of cytokines and chemokines known to attract and activate key players of the innate and adaptive immune system. CONCLUSION: This translational approach not only allowed for identification of the basic mechanisms of self-adjuvantation from the mRNA vaccine but also for comparison of the response across species, a response that appears relatively conserved or at least convergent between the in vitro human and in vivo mouse models.


Assuntos
Adjuvantes Imunológicos/farmacologia , Engenharia Genética , Imunidade Inata/efeitos dos fármacos , Vacinas contra Influenza/imunologia , RNA Mensageiro/administração & dosagem , Pesquisa Translacional Biomédica , Animais , Sequência de Bases , Relação Dose-Resposta Imunológica , Regulação da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Imunidade Inata/genética , Linfonodos/metabolismo , Camundongos Endogâmicos C57BL
7.
Mol Ther ; 23(9): 1456-64, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26050989

RESUMO

Being a transient carrier of genetic information, mRNA could be a versatile, flexible, and safe means for protein therapies. While recent findings highlight the enormous therapeutic potential of mRNA, evidence that mRNA-based protein therapies are feasible beyond small animals such as mice is still lacking. Previous studies imply that mRNA therapeutics require chemical nucleoside modifications to obtain sufficient protein expression and avoid activation of the innate immune system. Here we show that chemically unmodified mRNA can achieve those goals as well by applying sequence-engineered molecules. Using erythropoietin (EPO) driven production of red blood cells as the biological model, engineered Epo mRNA elicited meaningful physiological responses from mice to nonhuman primates. Even in pigs of about 20 kg in weight, a single adequate dose of engineered mRNA encapsulated in lipid nanoparticles (LNPs) induced high systemic Epo levels and strong physiological effects. Our results demonstrate that sequence-engineered mRNA has the potential to revolutionize human protein therapies.


Assuntos
Expressão Gênica , Terapia Genética , RNA Mensageiro/genética , Animais , Linhagem Celular , Índices de Eritrócitos , Eritropoetina/sangue , Eritropoetina/genética , Eritropoetina/metabolismo , Genes Reporter , Terapia Genética/métodos , Humanos , Lipídeos/química , Macaca fascicularis , Camundongos , Modelos Animais , Nanopartículas/química , RNA Mensageiro/administração & dosagem , RNA Mensageiro/química , Mutação Silenciosa , Sus scrofa
8.
Int J Cancer ; 137(2): 372-84, 2015 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-25530186

RESUMO

Protein- and peptide-based tumor vaccines depend on strong adjuvants to induce potent immune responses. Here, we demonstrated that a recently developed novel adjuvant based on a non-coding, long-chain RNA molecule, termed RNAdjuvant(®) , profoundly increased immunogenicity of both antigen formats. RNAdjuvant(®) induced balanced, long-lasting immune responses that resulted in a strong anti-tumor activity. A direct comparison to Poly(I:C) showed superior efficacy of our adjuvant to enhance antigen-specific multifunctional CD8(+) T-cell responses and mediate anti-tumor responses induced by peptide derived from HPV-16 E7 protein in the syngeneic TC-1 tumor, a murine model of human HPV-induced cervical cancer. Moreover, the adjuvant was able to induce functional memory responses that mediated complete tumor remission. Despite its remarkable immunostimulatory activity, our RNA-based adjuvant exhibited an excellent pre-clinical safety profile. It acted only locally at the injection site where it elicited a transient but strong up-regulation of pro-inflammatory and anti-viral cytokines as well as cytoplasmic RNA sensors without systemic cytokine release. This was followed by the activation of immune cells in the draining lymph nodes. Our data indicate that our RNA-based adjuvant is a safe and potent immunostimulator that may profoundly improve the efficacy of a variety of cancer vaccines.


Assuntos
Adjuvantes Imunológicos , Vacinas Anticâncer/imunologia , RNA Longo não Codificante/imunologia , Neoplasias do Colo do Útero/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Vacinas Anticâncer/farmacologia , Linhagem Celular Transformada , Citocinas/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Humanos , Memória Imunológica/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas E7 de Papillomavirus/imunologia , Peptídeos/imunologia , Peptídeos/farmacologia , Poli I-C/imunologia , Poli I-C/farmacologia , RNA Longo não Codificante/genética , Resultado do Tratamento , Neoplasias do Colo do Útero/tratamento farmacológico
9.
BMC Cancer ; 14: 748, 2014 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-25288198

RESUMO

BACKGROUND: Advanced non-small cell lung cancer (NSCLC) represents a significant unmet medical need. Despite advances with targeted therapies in a small subset of patients, fewer than 20% of patients survive for more than two years after diagnosis. Cancer vaccines are a promising therapeutic approach that offers the potential for durable responses through the engagement of the patient's own immune system. CV9202 is a self-adjuvanting mRNA vaccine that targets six antigens commonly expressed in NSCLC (NY-ESO-1, MAGEC1, MAGEC2, 5 T4, survivin, and MUC1). METHODS/DESIGN: The trial will assess the safety and tolerability of CV9202 vaccination combined with local radiation designed to enhance immune responses and will include patients with stage IV NSCLC and a response or stable disease after first-line chemotherapy or therapy with an EGFR tyrosine kinase inhibitor. Three histological and molecular subtypes of NSCLC will be investigated (squamous and non-squamous cell with/without EGFR mutations). All patients will receive two initial vaccinations with CV9202 prior to local radiotherapy (5 GY per day for four successive days) followed by further vaccinations until disease progression. The primary endpoint of the study is the number of patients experiencing Grade >3 treatment-related adverse events. Pharmacodynamic analyses include the assessment of immune responses to the antigens encoded by CV9202 and others not included in the panel (antigen spreading) and standard efficacy assessments. DISCUSSION: RNActive self-adjuvanted mRNA vaccines offer the potential for simultaneously inducing immune responses to a wide panel of antigens commonly expressed in tumors. This trial will assess the feasibility of this approach in combination with local radiotherapy in NSCLC patients. TRIAL REGISTRATION: Clinicaltrials.gov: NCT01915524/EudraCT No.: 2012-004230-41.


Assuntos
Vacinas Anticâncer/genética , Carcinoma Pulmonar de Células não Pequenas/terapia , Neoplasias Pulmonares/terapia , RNA Mensageiro/administração & dosagem , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/efeitos adversos , Vacinas Anticâncer/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Quimioterapia Adjuvante , Terapia Combinada , Humanos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , RNA Mensageiro/efeitos adversos , RNA Mensageiro/uso terapêutico , Doses de Radiação , Radioterapia , Resultado do Tratamento
10.
J Gene Med ; 14(6): 428-39, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22262664

RESUMO

Direct vaccination with mRNA encoding tumor antigens is a novel and promising approach in cancer immunotherapy. CureVac's mRNA vaccines contain free and protamine-complexed mRNA. Such two-component mRNA vaccines support both antigen expression and immune stimulation. These self-adjuvanting RNA vaccines, administered intradermally without any additional adjuvant, induce a comprehensive balanced immune response, comprising antigen specific CD4+ T cells, CD8+ T cells and B cells. The balanced immune response results in a strong anti-tumor effect and complete protection against antigen positive tumor cells. This tumor inhibition elicited by mRNA vaccines is a result of the concerted action of different players. After just two intradermal vaccinations, we observe multiple changes at the tumor site, including the up-regulation of many genes connected to T and natural killer cell activation, as well as genes responsible for improved infiltration of immune cells into the tumor via chemotaxis. The two-component mRNA vaccines induce a very fast and boostable immune response. Therefore, the vaccination schedules can be adjusted to suit the clinical situation. Moreover, by combining the mRNA vaccines with therapies in clinical use (chemotherapy or anti-CTLA-4 antibody therapy), an even more effective anti-tumor response can be elicited. The first clinical data obtained from two separate Phase I/IIa trials conducted in PCA (prostate cancer) and NSCLC (non-small cell lung carcinoma) patients have shown that the two-component mRNA vaccines are safe, well tolerated and highly immunogenic in humans.


Assuntos
Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Neoplasias da Próstata/terapia , Animais , Antígenos de Neoplasias/genética , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/imunologia , Linhagem Celular , Terapia Combinada , Células HeLa , Humanos , Imunoterapia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias da Próstata/genética , Neoplasias da Próstata/imunologia , RNA Mensageiro/genética , Vacinas de DNA
11.
RNA Biol ; 9(11): 1319-30, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23064118

RESUMO

mRNA vaccines combine desirable immunological properties with an outstanding safety profile and the unmet flexibility of genetic vaccines. Based on in situ protein expression, mRNA vaccines are capable of inducing a balanced immune response comprising both cellular and humoral immunity while not subject to MHC haplotype restriction. In addition, mRNA is an intrinsically safe vector as it is a minimal and only transient carrier of information that does not interact with the genome. Because any protein can be expressed from mRNA without the need to adjust the production process, mRNA vaccines also offer maximum flexibility with respect to development. Taken together, mRNA presents a promising vector that may well become the basis of a game-changing vaccine technology platform. Here, we outline the current knowledge regarding different aspects that should be considered when developing an mRNA-based vaccine technology.


Assuntos
Adjuvantes Imunológicos , RNA Mensageiro/imunologia , Vacinas Sintéticas/imunologia , Animais , Transporte Biológico , Expressão Gênica , Humanos , RNA Mensageiro/administração & dosagem , RNA Mensageiro/metabolismo
12.
RNA Biol ; 8(4): 627-36, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21654214

RESUMO

Insertional mutagenesis and the inherent risk of malignancy compromise the clinical use of DNA-based therapies. Being a transient copy of genetic material, mRNA is a safe alternative, overcoming this limitation. As a prerequisite for the development of efficient mRNA-based therapies, we investigated the cellular uptake and intracellular fate of mRNA for the first time. To this end we determined cell-type, dose and energy dependence of mRNA internalisation. Moreover, we employed markers for uptake pathways and cellular compartments to analyse the route of mRNA internalisation and its intracellular destination. Finally, we addressed the involvement of receptors and their nature using a competitor-based approach. We found that all cell types tested were amenable to uptake and expression of naked mRNA. Internalisation mainly occurred via caveolae/lipid raft-rich membrane domains and involved scavenger-receptor(s). Following endocytosis, mRNA eventually accumulated in lysosomes, while part of it escaped into the cytosol giving rise to protein synthesis. Taken together, our findings provide unprecedented insights into the internalisation and trafficking of exogenous mRNA, greatly facilitating the development of effective mRNA-based therapies in the future.


Assuntos
Endocitose , Lisossomos/metabolismo , RNA Mensageiro/metabolismo , Transporte Biológico , Carbocianinas , Cavéolas/metabolismo , Células HEK293 , Células HeLa , Humanos , Microdomínios da Membrana/metabolismo , Microscopia de Fluorescência , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , Receptores Depuradores/metabolismo
13.
NPJ Vaccines ; 6(1): 57, 2021 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-33863911

RESUMO

mRNA technologies have recently proven clinical efficacy against coronavirus disease 2019 and are among the most promising technologies to address the current pandemic. Here, we show preclinical data for our clinical candidate CVnCoV, a lipid nanoparticle-encapsulated mRNA vaccine that encodes full-length, pre-fusion stabilised severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein. In contrast to previously published approaches, CVnCoV is exclusively composed of naturally occurring nucleotides. Immunisation with CVnCoV induced strong humoral responses with high titres of virus-neutralising antibodies and robust T-cell responses. CVnCoV vaccination protected hamsters from challenge with wild-type SARS-CoV-2, demonstrated by the absence of viral replication in the lungs. Hamsters vaccinated with a suboptimal dose of CVnCoV leading to breakthrough viral replication exhibited no evidence of vaccine-enhanced disease. Overall, data presented here provide evidence that CVnCoV represents a potent and safe vaccine candidate against SARS-CoV-2.

14.
Wien Klin Wochenschr ; 133(17-18): 931-941, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34378087

RESUMO

BACKGROUND: We used the RNActive® technology platform (CureVac N.V., Tübingen, Germany) to prepare CVnCoV, a COVID-19 vaccine containing sequence-optimized mRNA coding for a stabilized form of SARS-CoV­2 spike (S) protein encapsulated in lipid nanoparticles (LNP). METHODS: This is an interim analysis of a dosage escalation phase 1 study in healthy 18-60-year-old volunteers in Hannover, Munich and Tübingen, Germany, and Ghent, Belgium. After giving 2 intramuscular doses of CVnCoV or placebo 28 days apart we assessed solicited local and systemic adverse events (AE) for 7 days and unsolicited AEs for 28 days after each vaccination. Immunogenicity was measured as enzyme-linked immunosorbent assay (ELISA) IgG antibodies to SARS-CoV­2 S­protein and receptor binding domain (RBD), and SARS-CoV­2 neutralizing titers (MN50). RESULTS: In 245 volunteers who received 2 CVnCoV vaccinations (2 µg, n = 47, 4 µg, n = 48, 6 µg, n = 46, 8 µg, n = 44, 12 µg, n = 28) or placebo (n = 32) there were no vaccine-related serious AEs. Dosage-dependent increases in frequency and severity of solicited systemic AEs, and to a lesser extent local AEs, were mainly mild or moderate and transient in duration. Dosage-dependent increases in IgG antibodies to S­protein and RBD and MN50 were evident in all groups 2 weeks after the second dose when 100% (23/23) seroconverted to S­protein or RBD, and 83% (19/23) seroconverted for MN50 in the 12 µg group. Responses to 12 µg were comparable to those observed in convalescent sera from known COVID-19 patients. CONCLUSION: In this study 2 CVnCoV doses were safe, with acceptable reactogenicity and 12 µg dosages elicited levels of immune responses that overlapped those observed in convalescent sera.


Assuntos
COVID-19 , Nanopartículas , Vacinas , Adolescente , Adulto , Anticorpos Antivirais , COVID-19/terapia , Vacinas contra COVID-19 , Método Duplo-Cego , Humanos , Imunização Passiva , Imunogenicidade da Vacina , Lipídeos , Pessoa de Meia-Idade , RNA Mensageiro , SARS-CoV-2 , Adulto Jovem , Soroterapia para COVID-19
15.
Vaccine ; 37(13): 1819-1826, 2019 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-30797640

RESUMO

BACKGROUND: We report the first-in-concept human trial of the safety, tolerability and immunogenicity when a novel TLR 7/8/RIG I agonist RNA-based adjuvant, CV8102, was administered alone or mixed with fractional doses of a licensed rabies vaccine (Rabipur®) as model antigen. METHODS: The primary objective was to assess the safety and reactogenicity of various dose levels of CV8102 alone or mixed with Rabipur® in healthy 18-40 year-old male volunteers. A secondary objective was to assess the immune-enhancing potential of bedside-mixes of CV8102 with fractional doses of Rabipur® by measuring induction of rabies virus neutralising titres (VNTs). RESULTS: Fifty-six volunteers received 50-100 µg CV8102 alone (n = 11), bedside-mixed CV8102 and Rabipur® (n = 20), or Rabipur® alone (n = 25; control). When given alone or mixed with Rabipur® CV8102 caused mostly Grade 1 or 2 local or systemic reactogenicity, but no related SAEs. As 100 µg CV8102 was associated with marked CRP increases further dose escalation was stopped. Combining 25-50 µg of CV8102 with fractional doses of Rabipur® significantly improved the kinetics of VNT responses; 50 µg CV8102 also improved the magnitude of VNT responses to 1/10 Rabipur® but caused severe but self-limiting influenza-like symptoms in 2 of 14 subjects. CONCLUSIONS: Doses of 25 and 50 µg CV8102 appeared safe and with an acceptable reactogenicity profile while significantly enhancing the immunogenicity of fractional doses of rabies vaccine. EudraCT No. 2013-004514-18.


Assuntos
Adjuvantes Imunológicos , Imunogenicidade da Vacina , Vacina Antirrábica/imunologia , Vírus da Raiva/imunologia , Raiva/imunologia , Raiva/prevenção & controle , Adulto , Anticorpos Neutralizantes , Anticorpos Antivirais , Voluntários Saudáveis , Humanos , Esquemas de Imunização , Masculino , Avaliação de Resultados em Cuidados de Saúde , Vacina Antirrábica/administração & dosagem , Vacina Antirrábica/efeitos adversos , Vacinação , Adulto Jovem
16.
J Immunother Cancer ; 7(1): 38, 2019 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-30736848

RESUMO

BACKGROUND: Preclinical studies demonstrate synergism between cancer immunotherapy and local radiation, enhancing anti-tumor effects and promoting immune responses. BI1361849 (CV9202) is an active cancer immunotherapeutic comprising protamine-formulated, sequence-optimized mRNA encoding six non-small cell lung cancer (NSCLC)-associated antigens (NY-ESO-1, MAGE-C1, MAGE-C2, survivin, 5T4, and MUC-1), intended to induce targeted immune responses. METHODS: We describe a phase Ib clinical trial evaluating treatment with BI1361849 combined with local radiation in 26 stage IV NSCLC patients with partial response (PR)/stable disease (SD) after standard first-line therapy. Patients were stratified into three strata (1: non-squamous NSCLC, no epidermal growth factor receptor (EGFR) mutation, PR/SD after ≥4 cycles of platinum- and pemetrexed-based treatment [n = 16]; 2: squamous NSCLC, PR/SD after ≥4 cycles of platinum-based and non-platinum compound treatment [n = 8]; 3: non-squamous NSCLC, EGFR mutation, PR/SD after ≥3 and ≤ 6 months EGFR-tyrosine kinase inhibitor (TKI) treatment [n = 2]). Patients received intradermal BI1361849, local radiation (4 × 5 Gy), then BI1361849 until disease progression. Strata 1 and 3 also had maintenance pemetrexed or continued EGFR-TKI therapy, respectively. The primary endpoint was evaluation of safety; secondary objectives included assessment of clinical efficacy (every 6 weeks during treatment) and of immune response (on Days 1 [baseline], 19 and 61). RESULTS: Study treatment was well tolerated; injection site reactions and flu-like symptoms were the most common BI1361849-related adverse events. Three patients had grade 3 BI1361849-related adverse events (fatigue, pyrexia); there was one grade 3 radiation-related event (dysphagia). In comparison to baseline, immunomonitoring revealed increased BI1361849 antigen-specific immune responses in the majority of patients (84%), whereby antigen-specific antibody levels were increased in 80% and functional T cells in 40% of patients, and involvement of multiple antigen specificities was evident in 52% of patients. One patient had a partial response in combination with pemetrexed maintenance, and 46.2% achieved stable disease as best overall response. Best overall response was SD in 57.7% for target lesions. CONCLUSION: The results support further investigation of mRNA-based immunotherapy in NSCLC including combinations with immune checkpoint inhibitors. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT01915524 .


Assuntos
Adjuvantes Imunológicos/uso terapêutico , Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/terapia , Imunoterapia , Neoplasias Pulmonares/radioterapia , Neoplasias Pulmonares/terapia , Pemetrexede/uso terapêutico , Protaminas/uso terapêutico , RNA Mensageiro/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Neoplasias/genética , Carcinoma Pulmonar de Células não Pequenas/imunologia , Terapia Combinada , Feminino , Humanos , Neoplasias Pulmonares/imunologia , Masculino , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Mucina-1/genética , Proteínas de Neoplasias/genética , Survivina/genética
17.
NPJ Vaccines ; 2: 29, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29263884

RESUMO

mRNA represents a promising new vaccine technology platform with high flexibility in regard to development and production. Here, we demonstrate that vaccines based on sequence optimized, chemically unmodified mRNA formulated in optimized lipid nanoparticles (LNPs) are highly immunogenic and well tolerated in non-human primates (NHPs). Single intramuscular vaccination of NHPs with LNP-formulated mRNAs encoding rabies or influenza antigens induced protective antibody titers, which could be boosted and remained stable during an observation period of up to 1 year. First mechanistic insights into the mode of action of the LNP-formulated mRNA vaccines demonstrated a strong activation of the innate immune response at the injection site and in the draining lymph nodes (dLNs). Activation of the innate immune system was reflected by a transient induction of pro-inflammatory cytokines and chemokines and activation of the majority of immune cells in the dLNs. Notably, our data demonstrate that mRNA vaccines can compete with licensed vaccines based on inactivated virus or are even superior in respect of functional antibody and T cell responses. Importantly, we show that the developed LNP-formulated mRNA vaccines can be used as a vaccination platform allowing multiple, sequential vaccinations against different pathogens. These results provide strong evidence that the mRNA technology is a valid approach for the development of effective prophylactic vaccines to prevent infectious diseases.

18.
EMBO Mol Med ; 9(10): 1434-1447, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28794134

RESUMO

The delivery of genetic information has emerged as a valid therapeutic approach. Various reports have demonstrated that mRNA, besides its remarkable potential as vaccine, can also promote expression without inducing an adverse immune response against the encoded protein. In the current study, we set out to explore whether our technology based on chemically unmodified mRNA is suitable for passive immunization. To this end, various antibodies using different designs were expressed and characterized in vitro and in vivo in the fields of viral infections, toxin exposure, and cancer immunotherapies. Single injections of mRNA-lipid nanoparticle (LNP) were sufficient to establish rapid, strong, and long-lasting serum antibody titers in vivo, thereby enabling both prophylactic and therapeutic protection against lethal rabies infection or botulinum intoxication. Moreover, therapeutic mRNA-mediated antibody expression allowed mice to survive an otherwise lethal tumor challenge. In conclusion, the present study demonstrates the utility of formulated mRNA as a potent novel technology for passive immunization.


Assuntos
Antitoxina Botulínica/imunologia , Botulismo/prevenção & controle , Imunização Passiva/métodos , Profilaxia Pós-Exposição , RNA Mensageiro/administração & dosagem , Vacina Antirrábica/imunologia , Raiva/prevenção & controle , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Antitoxina Botulínica/administração & dosagem , Antitoxina Botulínica/sangue , Botulismo/terapia , Relação Dose-Resposta Imunológica , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Raiva/terapia , Vacina Antirrábica/administração & dosagem , Vacina Antirrábica/sangue , Vírus da Raiva/imunologia
19.
Vaccine ; 34(33): 3882-93, 2016 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-27269061

RESUMO

mRNA represents a new platform for the development of therapeutic and prophylactic vaccines with high flexibility with respect to production and application. We have previously shown that our two component self-adjuvanted mRNA-based vaccines (termed RNActive® vaccines) induce balanced immune responses comprising both humoral and cellular effector as well as memory responses. Here, we evaluated the early events upon intradermal application to gain more detailed insights into the underlying mode of action of our mRNA-based vaccine. We showed that the vaccine is taken up in the skin by both non-leukocytic and leukocytic cells, the latter being mostly represented by antigen presenting cells (APCs). mRNA was then transported to the draining lymph nodes (dLNs) by migratory dendritic cells. Moreover, the encoded protein was expressed and efficiently presented by APCs within the dLNs as shown by T cell proliferation and immune cell activation, followed by the induction of the adaptive immunity. Importantly, the immunostimulation was limited to the injection site and lymphoid organs as no proinflammatory cytokines were detected in the sera of the immunized mice indicating a favorable safety profile of the mRNA-based vaccines. Notably, a substantial boostability of the immune responses was observed, indicating that mRNA can be used effectively in repetitive immunization schedules. The evaluation of the immunostimulation following prime and boost vaccination revealed no signs of exhaustion as demonstrated by comparable levels of cytokine production at the injection site and immune cell activation within dLNs. In summary, our data provide mechanistic insight into the mode of action and a rational for the use of mRNA-based vaccines as a promising immunization platform.


Assuntos
Imunidade Adaptativa , Adjuvantes Imunológicos/administração & dosagem , Imunidade Inata , RNA Mensageiro/imunologia , Vacinas/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Citocinas/imunologia , Feminino , Injeções Intradérmicas , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL
20.
Oncoimmunology ; 5(12): e1249560, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-28123889

RESUMO

We recently completed a phase I/IIa trial of RNActive® CV9201, a novel mRNA-based therapeutic vaccine targeting five tumor-associated antigens in non-small cell lung cancer (NSCLC) patients. The aim of the study presented here was to comprehensively analyze changes in peripheral blood during the vaccination period and to generate hypotheses facilitating the identification of potential biomarkers correlating with differential clinical outcomes post RNActive® immunotherapy. We performed whole-genome expression profiling in a subgroup of 22 stage IV NSCLC patients before and after initiation of treatment with CV9201. Utilizing an analytic approach based on blood transcriptional modules (BTMs), a previously described, sensitive tool for blood transcriptome data analysis, patients segregated into two major clusters based on transcriptional changes post RNActive® treatment. The first group of patients was characterized by the upregulation of an expression signature associated with myeloid cells and inflammation, whereas the other group exhibited an expression signature associated with T and NK cells. Patients with an enrichment of T and NK cell modules after treatment compared to baseline exhibited significantly longer progression-free and overall survival compared to patients with an upregulation of myeloid cell and inflammatory modules. Notably, these gene expression signatures were mutually exclusive and inversely correlated. Furthermore, our findings correlated with phenotypic data derived by flow cytometry as well as the neutrophil-to-lymphocyte ratio. Our study thus demonstrates non-overlapping, distinct transcriptional profiles correlating with survival warranting further validation for the development of biomarker candidates for mRNA-based immunotherapy.

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